The hepatitis C virus (HCV) is a virus belonging to the family Flaviviridae. It has a single-stranded (+) strand sense RNA as its genome and is known to cause hepatitis C. Recent studies have revealed that Hepatitis C virus is classified into a number of types based on genotypes or serotypes. According to the phylogenetic analysis of Simmonds et al., using the nucleotide sequences of the HCV strains, which is currently a mainstream method of classifying HCV genotypes, HCV is classified into 6 genotypes: genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a and genotype 3b (see Simmonds, P. et al, Hepatology, (1994) 10, pp. 1321-1324). Each of these types is further classified into several subtypes. The nucleotide sequences of the full-length genomes of a several number of genotypes of HCV have been determined to date (see JP Patent Publication (Kokai) No. 2002-171978 A; Choo et al., Science, (1989) 244, pp. 359-362; Kato et al., J. Med. Virol., (2001) 64(3) pp. 334-339; Okamoto, H et al, J. Gen, Virol., (1992) 73 pp. 673-679; and Mori, S. et al, Biochem. Biophis. Res. Commun., (1992) 183, pp. 334-342).
HCV causes chronic hepatitis by persistent infection. Currently, the main cause of chronic hepatitis observed worldwide is persistent HCV infection. Actually, around 50% of individuals with persistent infection develop chronic hepatitis. Chronic hepatitis in approximately 20% of these patients shifts to liver cirrhosis over the course of 10 to 20 years, and some of these patients further go on to advanced lethal pathological conditions such as hepatic cancer.
Hepatitis C is currently treated mainly by a therapy using interferon-α or interferon-β, or a therapy using in combination interferon-α and ribavirin, the purine-nucleoside derivative. However, even when these therapies are performed, the therapeutic effects are observed in only approximately 60% of all the treated patients. When the therapies are ceased after the exertion of the effects, the disease recrudesces in more than half of the patients. The therapeutic effect of interferones is known to relate to HCV genotypes, and is said to be lower against genotype 1b and higher against genotype 2a (see Yoshioka et al., Hepatology, (1992) 16(2): pp. 293-299).
It is an important goal to develop therapeutic agents or prophylactic agents effective against hepatitis C, the incidence rate of which is high in industrial countries, for which currently no causal treatment are present, and which finally bring about serious results. Hence, the development of HCV-specific chemotherapies and vaccine therapies are earnestly desired. A target for the development of an anti-HCV agent may be the suppression of HCV replication or the suppression of infection of cells with HCV.
Until recently, propagation of HCV in a cell culture system and infecting cultured cells with HCV have been difficult. Moreover, a chimpanzee has been the only animal that can be infected with HCV and can be used in experiments, so that it has been difficult to carry out studies on the replication mechanism of HCV and the infection mechanism of HCV. However, recently, HCV subgenomic RNA replicons have been prepared as HCV-derived autonomously replicable RNA (see JP Patent Publication (Kokai) No. 2001-17187 A; Lohmann et al., Science, (1999) 285, pp. 110-113; Blight et al., Science, (2000) 290, pp. 1972-1974; Friebe et al., J. Viral., (2001) 75(24): pp. 12047-12057; Ikeda et al., J. Virol., (2002) 76(6): pp. 2997-3006), which enables the analysis of the replication mechanism of HCV using cultured cells. These HCV subgenomic RNA replicons are each prepared by substituting structural proteins existing downstream of HCV IRES in the 5′ untranslated region of the HCV genomic RNA of genotype 1b with a neomycin resistance gene and EMCV IRES that has been ligated downstream of the resistance gene. It has been demonstrated that this RNA replicon is autonomously replicated in human hepatic cancer cells, Huh7 cells, when introduced into the Huh7 cells followed by culture in the presence of neomycin.
However, regarding such intracellular RNA replication systems for HCV, only those using HCV genomic RNA of genotype 1b have been prepared so far. Since there has been a report that different genotypes of HCV differ also in viral proteins encoded, it may be difficult to sufficiently elucidate the replication mechanism of HCV only by analyzing the subgenomic RNA replicons derived from HCV of genotype 1b. Furthermore, based on the fact that the therapeutic effects of interferons differ depending on the HCV genotypes, it may be particularly difficult to develop an anti-HCV agent having an effect on various types of HCV by the use of only an HCV replication system containing the subgenomic RNA replicon of HCV of genotype 1b.